Identification, design, and validation of a neoantigen-based peptide vaccine for the treatment of PAX3 / PAX7-FKHR fusion-positive alveolar rhabdomyosarcoma

Abstract

In some aspects there is described formulations for the treatment of fusion-positive alveolar rhabdomyosarcoma, methods of preparing thereof, and validating their immunogenic activity.

Description

IDENTIFICATION, DESIGN, AND VALIDATION OF A NEOANTIGEN-BASED PEPTIDE VACCINE FOR THE TREATMENT OF PAX3 / PAX7-FKHR FUSION¬POSITIVE ALVEOLAR RHABDOMYOSARCOMAFIELD OF THE INVENTION

[0001] In some aspects, there is described formulations for the treatment of fusion-positive alveolar rhabdomyosarcoma, methods of preparing thereof, and validating their immunogenic activity.BACKGROUND

[0002] Rhabdomyosarcoma (RMS) is a malignant disease of cancer cells forming a tumor in muscle tissue. It is the most common pediatric soft tissue sarcoma. There are two main types of RMS, embryonal and alveolar, in addition to less common types that vary in incidence by age, location of tumor, and prognosis. Alveolar RMS (ARMS) is generally more aggressive, affects all ages equally but constitutes a larger portion of RMS in older children and adults, and primarily occurs in the torso, arms, and legs. Although there is no known cause for most cases of RMS, risk factors may include the following genetic conditions: Li- Fraumeni syndrome, Dicerl syndrome, Neurofibromatosis type 1, Costello syndrome, Beckwith-Wiedemann syndrome, and Noonan syndrome. The staging / grouping system is based on the size and location of the tumor and whether it has metastasized and spread to other tissues. Based on the stage / group, high-risk RMS has an extremely poor prognosis, with a 5-year observed survival of -25%. Treatment options for RMS include surgery, radiation, and chemotherapy. This standard of care is associated with significant short- and long-term toxicities and a poor quality-of-life, especially in young children. Additionally, the cancer may sometimes relapse and return or become refractory and resistant to current therapies. Hence, there is an urgent, unmet need to develop novel therapeutic approaches that are less toxic for the treatment of RMS and to prevent relapse in patients with high-risk disease.

[0003] Cancer cells typically generate mutant proteins that are not found in normal, healthy cells. These new proteins represent neoantigens that may stimulate an immune response and be targets for novel therapies. Forkhead box protein 01 (FoxOl), also known as forkhead in RMS (FKHR), is a transcription factor that frequently forms a novel fusion with paired box gene 3 (Pax3) or paired box gene 7 (Pax7) transcription factors due to chromosomal translocations. These Pax3-FKHR and Pax7-FKHR fusions are found in 60% and 24% cases of ARMS, respectively. The fusion genes encode chimeric proteins exhibiting deregulated transcription and subsequent oncogenic activity. Several studies have associated fusion statuswith poor prognosis. This distinct molecule provides a viable opportunity to specifically target malignant cells as peptides derived from fusion proteins are not found in normal tissue.

[0004] The major histocompatibility complex (MHC) binds pathogen- and self-derived peptides to display them on the cell’s surface for recognition by T-cells. The MHC includes class I and class II human leukocyte antigen (HLA) genes that are highly polymorphic across the population, which results in a wide range of different peptide-binding specificities. MHC class I is ubiquitously expressed in all nucleated cells and binds peptides generated from proteosome-mediated degradation of cytosolic proteins that are transported to the endoplasmic reticulum by the transporter associated with antigen processing (TAP). Ultimately, the peptide-bound complex is externally displayed on the surface of the cell for cytotoxic CD8+ T-cells. Although MHC class I primarily binds intracellular self (for autorecognition or autoimmune) or foreign (such as in virus-infected cells) peptide, it also may present exogenous peptides in a process known as cross-presentation. The optimal length for MHC class I peptides is nine amino acids (9-mer) but binding has been demonstrated for 8-14 amino acid peptides. MHC class II are only expressed in professional antigen-presenting cells (APCs), such as dendritic cells and B-cells, and primarily bind exogenous peptides acquired through phagocytosis for presentation to CD4+ helper T-cells. The length of peptides bound by MHC class II can vary considerably, generally 13-25 amino acids. Finally, activated T-cells release cytokines such as interferon-gamma (IFN-y) to regulate the immune response. Various strategies to modulate the immune system have recently been explored as novel cancer therapeutics. As a proof of concept, recent clinical trials have demonstrated neoantigen-mediated T-cell-specific responses, providing rationale for selection of tumorspecific neoantigens to generate specific and durable immunotherapies.BRIEF SUMMARY

[0005] In one aspect there is provided an isolated polypeptide that comprises or consists of: an amino acid sequence having at least 90% identity with an amino acid sequence set forth in the following table:

[0006] In one aspect there is provided an isolated polypeptide that comprises or consists of: an amino acid sequence having an amino acid sequence set forth in the following table:

[0007] In one aspect there is provided an isolated nucleic acid molecule comprising a nucleotide sequence encoding a peptide comprising or consisting of an amino acid sequence having at least about 90% identity with the amino acid sequence set forth in in the following table:

[0008] In one aspect there is provided an isolated nucleic acid molecule comprising a nucleotide sequence encoding a peptide comprising or consisting of an amino acid sequence having 100% identity with the amino acid sequence set forth in in the following table:

[0009] In one aspect there is provided a vector comprising the polynucleotide of claims 3 or 4.

[0010] In one aspect there is provided a mammalian cell comprising the polynucleotide of claim 3 or 4, or the vector of claim 5.

[0011] In one example, said mammalian cell is a human cell or non-human primate cell.

[0012] In one aspect there is provided a host cell comprising the polynucleotide of claim 3 or 4, or the vector of claim 5.

[0013] In one example, wherein said host cell is a mammalian cell, an insect cell, a bacteria cell, or a fungal cell.

[0014] In one aspect there is provided an antibody that specifically recognizes the polypeptide of claims 1 or 2.

[0015] In one example, wherein said antibody is a monoclonal antibody or a polyclonal antibody.

[0016] In one aspect there is provided a population of autologous dendritic cells or antigen presenting cells that have been pulsed with one or more of the peptides as defined in claim 1 or 2, or transfected with a polynucleotide encoding one or more of the polypeptides as defined in claim 3 or 4.

[0017] In one aspect there is provided a vaccine or immunogenic composition capable of raising a specific T-cell response comprising:

[0018] i) one or more polypeptide as defined in claim 1 or 2, optionally with a physiologically acceptable buffer, carrier, or excipient, and / or optionally with an adjuvant or immunostimulant;

[0019] ii) one or more polynucleotides of claims 3 or 4, optionally linked to a heterologous regulatory control nucleotide sequence; and / or

[0020] iii) a population of autologous dendritic cells or antigen presenting cells, as defined in claim 12.

[0021] In one aspect there is provided a method of treating a subject having a cancer, or suspect of having a cancer, or at risk of developing a cancer, comprising: administering the vaccine of claim 12.

[0022] In one example, wherein the cancer is Rhabdomyosarcoma (RMS).

[0023] In one example, wherein the RMS is embryonal RMS or alveolar RMS.

[0024] In one example, wherein the subject is a human.

[0025] In one example, wherein the human is a pediatric human.

[0026] In one aspect there is provided a method of treating a subject having a cancer, or suspect of having a cancer, or at risk of developing a cancer, comprising: administering Useof the vaccine of claim 12 for treating a subject having a cancer, or suspect of having a cancer, or at risk of developing a cancer, or in the manufacture of a medicament for treating a subject having a cancer, or suspect of having a cancer, or at risk of developing a cancer.

[0027] In one example, the cancer is Rhabdomyosarcoma (RMS).

[0028] In one example, the RMS is embryonal RMS or alveolar RMS.

[0029] In one example, the subject is a human.

[0030] In one example, the human is a pediatric human.BRIEF DESCRIPTION OF THE FIGURES

[0031] FIG. 1 illustrates amino acid sequence alignment of chromosome translocation- mediated Pax3-FKHR and Pax7-FKHR gene fusions (indicated by arrow between the red QN).

[0032] FIG. 2 illustrates alignment of MHC class I peptides (1-1 to 1-8), MHC class II peptides (II-l to II-5), and long peptides (LP-1 and LP-2) with Pax3-FKHR and Pax7-FKHR (fusion site is indicated with the red QN). Predicted proteasome cleavage sites in are indicated by periods (•).

[0033] FIG. 3 illustrates CD8+ T-cells from healthy donors (HD1 to HD7) and RMS patients (RMS1 to RMS3) were primed with class I (1-1 to 1-8) and long peptides (LP-1 and LP-2), stimulated with Pax3-FKHR fusion-positive Rh41 (panel A), Rh30 (panel B), or cognate peptide (panel C), and subjected to ELISpot assay to quantify the mean delta value of IFN-y-secreting spot forming cells (SFC). Representative ELISpot images are shown (panel D).

[0034] FIG. 4 illustrates CD4+ (panel A) and CD8+ (panel B) T-cells from healthy donors (HD3 to HD7) and RMS patients (RMS1 to RMS3) were primed with class II peptides (II- 1 to II-5), stimulated with cognate peptide, and subjected to ELISpot assay to quantify the mean delta value of IFN-y-secreting spot forming cells (SFC). Representative ELISpot images are shown (panel C).

[0035] FIG. 5 illustrates (A) RMS cell lines, including fusion-negative RD and fusionpositive Rh30 and Rh41, and Ewing sarcoma SK-ES cells were immunoblotted with antibodies against FKHR and B-actin. (B) CD8+ T-cells from donors (HD4, RMS2, and RMS 3) primed with pooled MCH class I peptides were stimulated with fusion-positive cells (Rh30 or Rh41) or fusion-negative cells (SK-ES) and subjected to ELISpot assay to quantifythe mean delta value of IFN-y-secreting spot forming cells (SFC). (C) Representative ELISpot images are shown.

[0036] FIG. 6 illustrates (A) RMS cell lines (RD, Rh30, Rh41) and HEK293 cells transfected with empty vector or pcDNA3 carrying Pax3-FKHR were immunoblotted with antibodies against FKHR and B-actin. (B) CD8+ T-cells from healthy donors primed with pooled MCH class I or class II peptides or long peptides (LP-1 and LP-2) were stimulated with HEK293 cells carrying empty vector or expressing exogenous Pax3-FKHR fusion protein and subjected to ELISpot assay to quantify the mean delta value of IFN-y-secreting spot forming cells (SFC). (C) Representative ELISpot images are shown.

[0037] FIG. 7 illustrates CD8+ and CD4+ T-cells were stimulated with PHA or anti-CD3 and compared to T-cell, DC, or cancer (Rh30 and Rh41) cells alone by IFN-y ELISpot assay. Recombinant IFN-y was used as positive control.

[0038] FIG. 8 illustrates fibroblasts and tumor cells isolated from a primary tumor specimen of a patient with fusion-positive RMS, fusion-positive human Rh41 and mouse KTD203 RMS cells were immunoblotted with antibodies against FKHR, N-myc, and B- actin.

[0039] FIG. 9 illustrates pairwise amino acid sequence alignment between human (h) and mouse (m) Pax3-FKHR fusion using EMBOSS Needle. " | , :, and ." indicate identical, conserved, and semi-conserved residues, respectively. The fusion breakpoint is indicated in red font.

[0040] FIG. 10 illustrates (A) relative spleen area measured in mice carrying KTD203 orthotopic xenografts and treated with adjuvant control or MHC-I, MHC-II, or LP peptides emulsified in IFA adjuvant. (B) Splenocytes isolated from mice were stimulated with KTD203 tumor cells and subjected to IFN-y ELISpot assays. (C) Representative ELISpot images are shown.

[0041] FIG. 11 illustrates KTD203 tumor lysates immunoblotted with normal mouse serum or serum isolated from mice carrying KTD203 tumors treated adjuvant control or MHC-I, MHC-II, or LP peptides emulsified in IFA adjuvant.

[0042] FIG. 12 illustrates flow cytometry analysis of relative immune cell tumor infiltration including lymphocytes, CD3+TCRb+ and CD3-TCRb-, CD4+ and CD8+ T-cells, 4-1BB+ activation, PD-1+ checkpoint, myeloid and non-myeloid, B-cells, and NK / ILC1.DETAILED DESCRIPTION

[0043] We describe an immunotherapeutic approach where the patient’s immune system will be trained to recognize fusion-positive tumor cells. Specifically, we report a public neoantigen peptide-based approach for the treatment of Pax3- and Pax7-FKHR fusionpositive ARMS showing effective cancer- and epitope-specific CD8+ and CD4+ T-cell activity against ARMS cells.

[0044] In one aspect there is provided a method of treating a subject with cancer, at risk of developing cancer, or suspected of having a cancer.

[0045] The term “subject”, as used herein, refers to an animal, and can include, for example, domesticated animals, such as cats, dogs, etc., livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), laboratory animals (e.g., mouse, rabbit, rat, guinea pig, etc.), mammals, non-human mammals, primates, non-human primates, rodents, birds, reptiles, amphibians, fish, and any other animal. In a specific example, the subject is a human. In another specific example, the human is a pediatric human or an adult human. In a specific example, the subject is a pediatric human (also referred to as a child).

[0046] The term “cancer”, as used herein, refers to a variety of conditions caused by the abnormal, uncontrolled growth of cells. Cells capable of causing cancer, referred to as “cancer cells”, possess characteristic properties such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and / or certain typical morphological features. Cancer cells may be in the form of a tumor, but such cells may also exist alone within a subject, or may be a non-tumorigenic cancer cell. A cancer can be detected in any of a number of ways, including, but not limited to, detecting the presence of a tumor or tumors (e.g., by clinical or radiological means), examining cells within a tumor or from another biological sample (e.g., from a tissue biopsy), measuring blood markers indicative of cancer, and detecting a genotype indicative of a cancer. However, a negative result in one or more of the above detection methods does not necessarily indicate the absence of cancer, e.g., a patient who has exhibited a complete response to a cancer treatment may still have a cancer, as evidenced by a subsequent relapse.

[0047] In a specific example, the cancer is Rhabdomyosarcoma (RMS). In another example, the cancer is embryonal RMS. In another example, the cancer is alveolar RMS (ARMS).

[0048] The term “treatment”, “treat”, or “treating” as used herein, refers to obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state ofdisease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, diminishment of the reoccurrence of disease, and remission (whether partial or total), whether detectable or undetectable. "Treating" and "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment.

[0049] The term "amelioration" or "ameliorates" as used herein refers to a decrease, reduction or elimination of a condition, disease, disorder, or phenotype, including an abnormality or symptom.

[0050] The term "symptom" of a disease or disorder is any morbid phenomenon or departure from the normal in structure, function, or sensation, experienced by a subject and indicative of disease.

[0051] A "treatment regimen" as used herein refers to a combination of dosage, frequency of administration, or duration of treatment, with or without addition of a second medication, including chemotherapy or another immunotherapy such as immune checkpoint inhibitors.

[0052] For example, a subject with cancer may be treated to prevent progression or alternatively a subject in remission can be treated with a compound or composition described herein to prevent recurrence.

[0053] In one example, there is provided an isolated polypeptide that comprises or consists of: an amino acid sequence having at least 90% identity with an amino acid sequence set forth in the following table:

[0054] In one example, there is provided an isolated polypeptide that comprises or consists of: an amino acid sequence having an amino acid sequence set forth in the following table:

[0055] The term “isolated”, as used herein, refers to altered or removed from the natural state. For example, a polypeptide or nucleic acid naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or polypeptide can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

[0042] In some examples, there is provided a nucleotide sequence encoding a polypeptide as described herein.

[0056] Unless otherwise specified, a “nucleotide sequence encoding a polypeptide” (and the like) includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a polypeptide protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s).

[0057] The terms “peptide,” “polypeptide,” and “protein”, as used herein are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein's or peptide's sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

[0058] Further, the term "peptide or polypeptide," is used interchangeably with "neoantigenic peptide or polypeptide" in the present specification to designate a series of residues, typically L-amino acids, connected one to the other, typically by peptide bonds between the a-amino and carboxyl groups of adjacent amino acids. The polypeptides or peptides can be a variety of lengths, either in their neutral (uncharged) forms or in forms which are salts, and either free of modifications such as glycosylation, side chain oxidation, or phosphorylation or containing these modifications, subject to the condition that the modification does not destroy the biological activity of the polypeptides as herein described.

[0059] A tumor neoantigenic peptide is a peptide that arises from somatic alterations (classically mutations in the DNA sequence), is recognized as different from self, and is presented by antigen-presenting cells (APC), such as dendritic cells (DC) and tumor cells themselves. Cross- presentation plays an important role as the APC is able to translocate exogenous antigens from the phagosome into the cytosol for proteolytic cleavage into the major histocompatibility complex I (MHC I) epitopes by the proteasome.

[0060] A polypeptide as described herein may be obtained according to the methods of the present disclosure and thus encompasses one or more of the characteristics as described herein.

[0061] In some examples, the present disclosure provides ARMS tumor-specific neoantigen peptides, or a pool thereof, wherein said peptides:• are encoded by a part of an open reading frame (ORF) sequence from a transcript associated with the Pax3-FKHR or Pax7-FKHR fusion gene in a tumor sample;• comprises at least 9 amino acids and binds at least one MHC molecule with an affinity of less than 5000 nM or binds MHC class I or class II with a binding affinity of less than 10% percentile rank score predicted by NetMHCpan v4.1 (for MHC class I- restricted peptides) or NetMHCIIpan v4.0 (for MHC class Il-restricted peptides); and• are not expressed in normal, healthy cells.

[0062] According to an example of the present disclosure, the mutant peptides can notably be selected from ARMS and other fusion-positive malignancies including sarcomas such as biphenotypic sinonasal sarcoma.

[0063] The present disclosure also encompasses:• a population of autologous or allogeneic dendritic cells or antigen presenting cells that have been pulsed with one or more of the neoantigen peptides as herein defined, or transfected with a polynucleotide encoding one or more of such peptides;• a vaccine or immunogenic composition, notably a sterile vaccine or immunogenic composition, capable of raising a specific CD4+ and / or CD8+ T-cell response comprising: a. one or more neoantigen peptides as defined in the present disclosure, optionally wherein the neoantigen peptides are modified or combined into synthetic long peptides with cleavable linkers; b. one or more polynucleotides encoding a neoantigen peptide as herein defined; c. a population of autologous or allogeneic dendritic cells or antigen presenting cells that have been pulsed or loaded with one or more of the peptides as herein defined,

[0064] optionally in combination with a physiologically or pharmacologically acceptable buffer, carrier, excipient, immunostimulant and / or adjuvant.

[0065] The polypeptide herein may also be modified by extending or decreasing the compound's amino acid sequence, e.g., by the addition or deletion of amino acids. The peptides can also be modified by altering the order or composition of certain residues, it being readily appreciated that certain amino acid residues essential for biological activity, e.g., those at critical contact sites or conserved residues, may generally not be altered without an adverse effect on biological activity. The non-critical amino acids need not be limited to those naturally occurring in proteins, such as L-a-amino acids, or their D-isomers, but may include non-natural amino acids as well, such as b-g-d-amino acids, as well as many derivatives of L-a-amino acids.

[0066] Amino acid substitutions are typically of single residues. Substitutions, deletions, insertions or any combination thereof may be combined to arrive at a final peptide. Substitutional variants are those in which at least one residue of a peptide has been removed and a different residue inserted in its place.

[0067] The polypeptides herein may also comprise isosteres of two or more residues in the neoantigenic peptide or polypeptides. An isostere as defined here is a sequence of two or more residues that can be substituted for a second sequence because the steric conformation of the first sequence fits a binding site specific for the second sequence. The term specifically includes peptide backbone modifications well known to those skilled in the art. Such modifications include modifications of the amide nitrogen, the a-carbon, amide carbonyl, complete replacement of the amide bond, extensions, deletions or backbone crosslink.

[0068] Polypeptides may be made by any technique known to those of skill in the art, including the expression of proteins, polypeptides or peptides through standard molecular biological techniques, the isolation of proteins or peptides from natural sources, or the chemical synthesis of proteins or peptides.

[0069] Also provided herein is a vaccine or immunogenic composition capable of raising a specific T-cell response comprising: one or more polypeptides as described herein, one or more polynucleotides encoding a polypeptide as described herein; and / or a population of antigen presenting cells (such as autologous dendritic cells or artificial APC).

[0070] The vaccine or immunogenic composition may raise a specific cytotoxic T-cells response and / or a specific helper T-cell response.

[0071] The term “sequence identity” of a polypeptide or polynucleotide as used herein refers to a degree of sameness in an amino acid residue or a base in a specific region of two sequences that are aligned to best match each other for comparison. The sequence identity is a value obtained via alignment and comparison of the two sequences in the specific region for comparison, in which a partial sequence in the specific region for comparison may be added or deleted with respect to a reference sequence. The sequence identity represented in a percentage may be calculated by, for example, comparing two sequences that are aligned to best match each other in the specific region for comparison, determining matched sites with the same amino acid or base in the two sequences to obtain the number of the matched sites, dividing the number of the matched sites in the two sequences by a total number of sites in the compared specific regions (i.e., a size of the compared region), and multiplying a result of the division by 100 to obtain a sequence identity as a percentage. The sequenceidentity as a percentage may be determined using a known sequence comparison program, for example, BLASTP or BLASTN (NCBI), CLC Main Workbench (CLC bio), or MegAlign™ (DNASTAR Inc).

[0058] A polypeptide of may be synthesized by conventional techniques. For example, the peptides may be synthesized by chemical synthesis using solid phase peptide synthesis. These methods employ either solid or solution phase synthesis methods. Automated synthesis may be used.

[0072] In some example, a polypeptide may be produced by culturing a cell comprising a nucleic acid which encoded the polypeptide, and isolating the polypeptide from the host cell or culture medium thereof. The peptides of the invention may be post-translationally modified. For example, post-translational modifications that fall within the scope of the present invention include signal peptide cleavage, glycosylation, acetylation, isoprenylation, proteolysis, myristoylation, protein folding and proteolytic processing, etc. Some modifications or processing events require introduction of additional biological machinery. For example, processing events, such as signal peptide cleavage and core glycosylation, are examined by adding canine microsomal membranes or Xenopus egg extracts to a standard translation reaction.

[0073] In some examples, the polypeptides described herein may include unnatural amino acids formed by post-translational modification or by introducing unnatural amino acids during translation. A variety of approaches are available for introducing unnatural amino acids during protein translation.

[0074] A “cell” or “host cell” refers to an individual cell or cell culture that can be or has been a recipient of any recombinant vector(s), isolated polynucleotide, or polypeptide. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation and / or change. A host cell includes cells transfected or infected in vivo or in vitro with a recombinant vector or a polynucleotide of the invention. A host cell which comprises a recombinant vector of the invention is a recombinant host cell.

[0075] In one example, the host cell is a cell obtained or derived from a subject.

[0076] The term “pharmaceutically effective amount” as used herein refers to the amount of a drug or pharmaceutical agent that will elicit the biological or medical response of a tissue, system, animal or human that is being sought by a researcher or clinician. This amount can be a therapeutically effective amount.

[0077] Thus, as used herein, the term “therapeutically effective amount” refers to an amount that is effective for preventing, ameliorating, or treating a disease or disorder (e.g., cancer).

[0078] The term “pharmaceutically acceptable” as used herein refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0079] The term “pharmaceutically acceptable carrier” as used herein refers to a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject agents from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation, for example the carrier does not decrease the impact of the agent on the treatment. In other words, a carrier is pharmaceutically inert. The terms “physiologically tolerable carriers” and “biocompatible delivery vehicles” are used interchangeably. Thus, the term “carrier” or “excipient” may refer to a non-toxic solid, semi-solid or liquid filler, diluent. The term includes solvents, dispersion, media, coatings, isotonic agents, and adsorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art.

[0080] As used herein, the term “pharmaceutically-acceptable salts” refers to the conventional nontoxic salts or quaternary ammonium salt. These salts can be prepared in situ in the administration vehicle or the dosage form manufacturing process, or by separately reacting a compound in its free base or acid form with a suitable organic or inorganic acid or base, and isolating the salt thus formed during subsequent purification. Conventional nontoxic salts include those derived from inorganic acids such as sulfuric, sulfamic, phosphoric, nitric, and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, palmitic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicyclic, sulfanilic, 2- acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isothionic, and the like.

[0081] In some examples, therapeutic formulations comprising the compounds or compositions as described herein may be prepared for by mixing compounds or compositions having the desired degree of purity with optional physiologically acceptablecarriers, excipients or stabilizers, in the form of aqueous solutions, lyophilized or other dried formulations. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, histidine and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3- pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as TWEENTM, PLURONICSTM or polyethylene glycol (PEG).

[0082] A “pharmaceutical composition” as used herein refers to a chemical or biological composition suitable for administration to a subject. Such compositions may be specifically formulated for administration via one or more of a number of routes, including but not limited to, oral, parenteral, intravenous, intra-arterial, subcutaneous, intra-nasal, sublingual, intra-spinal, intra-cerebroventricular, and the like.

[0083] The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing the active compound into association with a carrier, which may constitute one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active compound with liquid carriers or finely divided solid carriers or both, and then if necessary, shaping the product.

[0084] The compounds and compositions may be administered to a subject by any convenient route of administration, whether systemically / peripherally or at the site of desired action, including but not limited to, oral (e.g. by ingestion); topical (including e.g. transdermal, intranasal, ocular, buccal, and sublingual); pulmonary (e.g. by inhalation or insufflation therapy using, e.g. an aerosol, e.g. through mouth or nose); rectal; vaginal; parenteral, for example, by injection, including subcutaneous, intradermal, intramuscular, intravenous, intra-arterial, intra-cardiac, intrathecal, intra-spinal, intra-capsular, sub- capsular, intra-orbital, intraperitoneal, intra-tracheal, subcuticular, intraarticular,subarachnoid, and intra- sternal; by implant of a depot / for example, subcutaneously or intramuscularly.

[0085] A skilled worker will be able to determine the appropriate dose for the individual subject by following the instructions on the label. Preparation and dosing schedules for commercially available second therapeutic and other compounds administered in combination with or concomitantly with compounds or compositions described herein may be used according to manufacturers' instructions or determined empirically by the skilled practitioner.

[0086] Method of the invention are conveniently practiced by providing the compounds and / or compositions used in such method in the form of a kit. Such a kit preferably contains the composition. Such a kit preferably contains instructions for the use thereof.

[0087] To gain a better understanding of the invention described herein, the following examples are set forth. It should be understood that these examples are for illustrative purposes only. Therefore, they should not limit the scope of this invention in anyway.EXAMPLES

[0088] METHODS

[0089] Identification of neoantigen peptides

[0090] Published papers were mined to estimate the frequency of fusion-positive RMS. The region of the Pax3-FKHR and Pax7-FKHR fusion site was constructed using sequences obtained from UniProt (Figure 1).

[0091] For MHC class I-mediated CD8+ T-cell responses, bioinformatic analyses utilizing artificial neural network (ANN)-trained MHC-binding, proteasomal cleavage, and cytotoxic T-cell epitope prediction models (NetMHC 4.0, NetCTL 1.2, NetMHCcons 1.1, and NetChop 3.1) were carried out using a 30 amino acid sequence with the fusion site located at position 15-16 to generate 9- to 14-mer neoantigen peptides with high binding affinity to the representative MHC class I supertype alleles: HLA-A*01 :01 (Al), HLA-A*02:01 (A2), HLA- A*03:01 (A3), HLA-A*24:02 (A24), HLA-A*26:01 (A26), HLA-B*07:02 (B7), HLA- A*08:01 (B8), HLA-B*27:05 (B27), HLA-B*39:01 (B39), HLA-B*40:01 (B44), HLA- B*58:01 (B58), and HLA-B*15:01 (B62). Candidates were selected from MHC class I neoantigen peptides that met the following criteria: (1) peptides containing the fusion site in the core sequence; (2) flanked by predicted cleavage sites of the human proteasome; and (3) preference for peptides with high binding affinity (<5% rank) or affinity to multiple, diverse MHC class I alleles (Table 1).

[0092] For MHC class Il-mediated CD4+ T-cell responses, ANN-trained MHC-binding prediction model (NetMHCII 2.3) was carried out using a 30 amino acid sequence with the fusion site located at position 15-16 to generate 15- to 18-mer neoantigen peptides with high binding affinity to the representative MHC class II alleles. Candidates were selected from MHC class II neoantigen peptides that met the following criteria: (1) peptides containing the fusion site in the core sequence; and (2) preference for peptides with high binding affinity (<5% rank) or affinity to multiple, diverse MHC class II alleles (Table 2).

[0093] Elongated 20- and 22-mer peptides containing the fusion site and previously identified candidates were then generated based on the potential for proteasomal processing into MHC class I neoantigens as predicted by the ANN-trained proteasome cleavage prediction model (Figure 2).

[0094] Peptide synthesis

[0095] Lyophilized neoantigen-derived peptides were synthesized at >95% purity by GenScript (Piscataway, NJ, USA) in a Good Manufacturing Practice (GMP) facility. Certificates of analysis, high performance liquid chromatography, mass spectrometry, and quantitative solubility testing reports are summarized in Table 3. Based on solubility testing, each peptide was dissolved to a stock concentration of 5 mg / ml in sterile, ultrapure water, aliquots stored at -80 °C, and thawed at room temperature before use.

[0096] Cell culture

[0097] Cells were cultured in complete RPMI-1640 media containing 10% FBS and maintained at 37 °C in a 5% CO2 humidified incubator. Pax3-FKHR fusion-positive ARMS cells, Rh30 and Rh41, and fusion-negative cancer cells, including RD (embryonal RMS), SK- ES (Ewing sarcoma), and HEK293 cells were used in the assays described below. Mouse rhabdomyosarcoma KTD203 cells carrying Pax3-FKHR fusion were used for in vivo xenograft models.

[0098] Isolation of dendritic cells and T-cells

[0099] Blood samples from normal, healthy donors (HD) and RMS patients in remission or at relapse (Table 4) were collected with informed consent and ethics board approval, including parental consent for pediatric patients. Table 4 lists characteristics of each donor used in this study. To confirm broad applicability, pediatric and adult male and female donors of different ethnicities were included to represent MHC diversity in the population. Peripheral blood mononuclear cells (PBMCs) were isolated from blood by density gradient centrifugation in Ficoll-Paque (Cytiva). Primary cells were used immediately or cryopreserved by initiallyfreezing at -80 °C in FBS containing 10% DMSO. These cells were used in ELISpot assays described below.

[0100] Monocytes were isolated from PBMCs by CD 14+ positive selection or the adherence method, in which monocytes attached to a culture flask for two hours with non-adherent cells removed by two washes in fresh media, followed by differentiation into monocyte-derived dendritic cells (Mo-DCs) in complete RPMI supplemented with 100 ng / ml granulocytemacrophage colony-stimulating factor (GM-CSF) and 25 ng / ml interleukin (IL)-4 for 5-7 days. Differentiated DCs (2,500 cells / well) were pulsed with 50 pg / ml peptide overnight for ~16 hours to generate mature DCs.

[0101] Cytotoxic CD8+ and helper CD4+ T-cells were isolated from PBMCs using CD8 and CD4 microbeads, respectively, and the MACS® magnetic cell separation method (Miltenyi Biotec). Following isolation, T-cells were co-cultured with neoantigen-pulsed DCs for five days (2,500 DCs and 25,000 T-cells / well; 1: 10 ratio of DC:T-cell) in complete RPMI supplemented with 10 ng / ml IL-2 and 10 ng / ml IL-7 for T-cell priming.

[0102] ELISpot assays

[0103] Primed T-cells (12,500-25,000 T-cells or 106splenocytes / well) were transferred to enzyme-linked immunosorbent spot (ELISpot) plates (R&D / Mabtech) and stimulated with cognate peptide (50 pg / ml) or Pax3-FKHR fusion-positive ARMS cancer cells (1 : 1 ratio of primed T-cells:cancer cells) to analyze secretion of IFN-y. Recombinant IFN-y (1.6 ng / well) and T-cells stimulated with phytohemagglutinin (PHA) lectin (0.25-2.5 pg / ml) or cluster of differentiation 3 (CD3) monoclonal antibody (1 pg / ml) were used as positive controls. Cells (Mo-DC, T-cells, and / or cancer cells) alone, unprimed T-cells with no peptide stimulated with cancer cells, or peptide-primed T-cells stimulated with fusion-negative cancer cells were used as controls. After incubation for 24 hours, ELISpot plates were developed according to the manufacturer’s protocol and imaged by dissection microscopy and S6 Universal M2 ELISpot reader (ImmunoSpot), followed by analysis and spot counting using ImmunoSpot v5.1.34 software. The baseline number of spots in negative controls were removed from test samples.

[0104] Plasmid DNA transfection

[0105] HEK293 cells were seeded 24 hours before transfection with pcDNA3 carrying empty vector or Pax3-FKHR by jetPRIME (1:2 ratio of pg DNA to pl transfection reagent) in complete media at 50% confluency for 24 hours.

[0106] Immunoblotting

[0107] Cell lysates in Laemmli sample buffer containing P-mercaptoethanol and sodium dodecyl sulfate were resolved by denaturing polyacrylamide gel electrophoresis. Proteinswere transferred to nitrocellulose membrane and immunoblotted with the indicated primary antibody (Santa Cruz Biotechnology). FKHR antibody detecting an epitope near the C- terminus of the protein was used to detect endogenous FKHR and Pax3-FKHR fusion protein and P-actin antibody was used to assess protein loading. Mouse serum was used to detect antibodies against tumor antigens. Following incubation with primary antibody for 1 hour, membranes were washed three times with tris buffered saline (TBS) containing 0.1% Tween- 20 and incubated with respective HRP-conjugated secondary antibody for 1 hour. Immunoreactive bands were detected using the Clarity ECL reagent. Western blots were imaged using the Bio-Rad ChemiDoc™ and analyzed using Image Lab software.

[0108] In vivo xenografts

[0109] All animal procedures were approved by the University of Calgary Animal Care Committee. C57BL / 6 mice (Charles River Laboratories) were housed with a 12: 12 h lightdark cycle at 23 °C and 40-60% relative humidity and provided with commercial rodent chow and water ad libitum. At day 0, six- to eight- week-old mice were injected into the rear leg muscle with 5.0 x 104KTD203 cells suspended in 0.1 mL PBS. After tumor cell injection, animals with detectable tumor growth were randomized into groups and treated with PBS vehicle control or neoantigen-based peptide vaccine. For vaccination, mice were treated once weekly with intramuscular injections of up to 100 pg of peptides emulsified in incomplete Freund's adjuvant (IFA). Mice in the vehicle control group received an equivalent volume of PBS in IFA via the same route of administration. Animals were monitored daily and their tumor areas were regularly estimated with a vernier caliper until any mouse met the endpoint criteria. Upon completion of the experiments, mice were euthanized followed by tumor mass measurements and isolation of serum and splenocytes for ex vivo assays. Single cell suspensions of tumors were prepared for flow cytometry analysis using an antibody panel against various immune cell populations including CD3e, CD19, CDl lb, CD45, CD4, CD8, PD-1, NKp46, TCRb, NK1.1, 4-1BB, and viability markers.

[0110] RESULTS

[0111] Validation of neoantigen peptides

[0112] Based on mutational analysis of RMS, neoantigen peptides containing the fusion site in Pax3-FKHR or Pax7-FKHR (Figure 1), which is found in over 80% of ARMS, were generated by bioinformatic analysis. Candidate peptides with high binding affinity to diverse MHC class I (Table 1) and class II (Table 2) alleles were selected for synthesis (Table 3). The Allele Frequency Net Database was used to estimate frequencies of these class I and II allelesto ensure the selected candidate peptides maximized coverage for the general population. Long peptides with potential for proteasome-mediated processing into any of the class I peptides were also generated (Figure 2).

[0113] To determine whether neoantigen peptides were effective at inducing MHC class I- mediated CD8+ and / or class Il-mediated CD4+ T-cell responses in vitro, IFN-y secretion was quantified by ELISpot assays. For this study, male and female individuals of diverse ethnicities were included to confirm efficacy across various MHC alleles (Table 4). Blood samples acquired from seven healthy, adult donors (HD1 to HD5) and three pediatric ARMS patients (RMS1 to RMS3) were processed into DCs and CD8+ or CD4+ T-cells. Differentiated DCs pulsed with neoantigen peptides were used to prime CD8+ or CD4+ T- cells.

[0114] Compared to no peptide control, class I peptides resulted in significant neoantigen- specific increases in IFN-y-secreting cells (Figure 3). Stimulation of primed CD8+ T-cells with fusion-positive ARMS cells, Rh41 (Figure 3A) or Rh30 (Figure 3B), was generally more effective than cognate peptide (Figure 3C), which may be explained by increased antigen presentation with the addition of cancer cells expressing MHC class I. As expected, each peptide displayed variable efficacy between individuals, likely due to variable affinities for different MHC alleles. Long peptides, which may be processed into numerous class I peptides, also stimulated strong CD8+ T-cell responses (Figure 3). Stimulation of class II peptide- primed CD4+ T-cells with cognate peptide increased IFN-y secretion (Figure 4A). Interestingly, class II peptides also exhibited responses in CD8+ T-cells (Figure 4B), indicating that these intermediate length peptides can stimulate both CD4+ and CD8+ T-cells through cross-presentation.

[0115] To test for specificity to fusion-positive cancer, CD8+ T-cells were primed with pooled class I peptides and stimulated with fusion-negative SK-ES cells derived from Ewing sarcoma (Figure 5A), which did not result in T-cell responses compared to fusion-positive ARMS cells (Figure 5B). Of note, SK-ES cells carry a different fusion protein, in which chromosome translocation between Ewing’s sarcoma breakpoint region 1 gene (EWSR1) and Friend leukemia virus integration site 1 gene (FLI1) form the EWSR1-FLI1 fusion gene commonly associated with Ewing sarcoma. In addition, HEK293 cells expressing exogenous Pax3-FKHR (Figure 6A) stimulated CD8+ T-cells primed with MHC class I, class II, or long peptides (Figure 6B).

[0116] As positive controls, T-cells were readily stimulated in a dose-dependent manner with PHA or anti-CD3, which are both potent activators of CD8+ and CD4+ T-cells, andrecombinant IFN-y confirmed antibody target specificity by the ELISpot assay (Figure 7). As negative controls, there was negligible IFN-y secretion in DCs, T-cells, and cancer cells alone.

[0117] In vivo validation of tumor-specific responses by neoantigen-based peptide vaccination in an immunocompetent mouse model is ongoing. Orthotopic xenograft tumors established in C57BL / 6 mice with KTD203 mouse cells, which express murine Pax3-FKHR that is identical to the antigenic fusion breakpoint region (Figures 8 and 9) and recapitulates various features of alveolar rhabdomyosarcomas in humans, were treated with adjuvant control or pooled peptides emulsified in IFA. Treatment with MHC-II and LP peptides resulted in enlargement of the spleen (Figure 10A), indicating proliferation of immune cells. Furthermore, compared to control, splenocytes isolated from mice exhibited increased IFN-y secretion when stimulated with KTD203 tumor cells (Figure 10B and 10C). Interestingly, MHC-II peptides did not respond to tumor cells, suggesting limited cross-presentation in vivo. To investigate antibody responses in vivo, serum isolated from mice carrying tumors was used to immunoblot KTD203 tumor lysates. Vaccination with MHC-II and LP peptides resulted in new immunoreactive bands compared to control (Figure 11), indicating antigen spreading, the phenomenon characterized by expansion of the immune response to secondary epitopes or antigens not directly targeted by the therapy. To explore immune cell infiltration into the tumor, single cell suspensions were prepared for flow cytometry analysis, which revealed vaccine-induced alterations in the immune landscape, notably increased activated T-cells, B-cells, and NK cells (Figure 12). However, high expression of PD-1 suggests combination with checkpoint inhibition may be necessary to overcome immunosuppressive mechanisms in the tumor microenvironment. Lastly, tumor growth properties and survival will be monitored.

[0118] Table 1 : MHC class I neoantigen peptide candidates identified with bioinformatic analysis.

[0119] Table 2 : MHC class II neoantigen peptide candidates identified with bioinformatic analysis.

[0120] Table 3: Physical and chemical properties of synthesized neoantigen peptides.

[0121] Table 4 : Characteristics of donor samples used in this study.

Claims

CLAIMSWhat is claimed is:

1. An isolated polypeptide that comprises or consists of: an amino acid sequence having at least 90% identity with an amino acid sequence set forth in the following table:

2. An isolated polypeptide that comprises or consists of: an amino acid sequence having an amino acid sequence set forth in the following table:

3. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a peptide comprising or consisting of an amino acid sequence having at least about 90% identity with the amino acid sequence set forth in in the following table:

4. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a peptide comprising or consisting of an amino acid sequence having 100% identity with the amino acid sequence set forth in in the following table:

5. A vector comprising the polynucleotide of claims 3 or 4.

6. A mammalian cell comprising the polynucleotide of claim 3 or 4, or the vector of claim 5.

7. The mammalian cell of claim 6, wherein said mammalian cell is a human cell or nonhuman primate cell.

8. A host cell comprising the polynucleotide of claim 3 or 4, or the vector of claim 5.

9. The host cell of claim 8, wherein said host cell is a mammalian cell, an insect cell, a bacteria cell, or a fungal cell.

10. An antibody that specifically recognizes the polypeptide of claims 1 or 2.

11. The antibody of claim 10, wherein said antibody is a monoclonal antibody or a polyclonal antibody.

12. A population of autologous dendritic cells or antigen presenting cells that have been pulsed with one or more of the peptides as defined in claim 1 or 2, or transfected with a polynucleotide encoding one or more of the polypeptides as defined in claim 3 or 4.

13. A vaccine or immunogenic composition capable of raising a specific T-cell response comprising:i) one or more polypeptide as defined in claim 1 or 2, optionally with a physiologically acceptable buffer, carrier, or excipient, and / or optionally with an adjuvant or immunostimulant; ii) one or more polynucleotides of claims 3 or 4, optionally linked to a heterologous regulatory control nucleotide sequence; and / or iii) a population of autologous dendritic cells or antigen presenting cells, as defined in claim 12.

14. A method of treating a subject having a cancer, or suspect of having a cancer, or at risk of developing a cancer, comprising: administering the vaccine of claim 12.

15. The method of claim 14, wherein the cancer is Rhabdomyosarcoma (RMS).

16. The method of claim 14 or 15, wherein the RMS is embryonal RMS or alveolar RMS.

17. The method of any one of claims 14 to 16, wherein the subject is a human.

18. The method of claim 17, wherein the human is a pediatric human.

19. A method of treating a subject having a cancer, or suspect of having a cancer, or at risk of developing a cancer, comprising: administering Use of the vaccine of claim 12 for treating a subject having a cancer, or suspect of having a cancer, or at risk of developing a cancer, or in the manufacture of a medicament for treating a subject having a cancer, or suspect of having a cancer, or at risk of developing a cancer.

20. The use of claim 19, wherein the cancer is Rhabdomyosarcoma (RMS).

21. The use of claim 19 or 20, wherein the RMS is embryonal RMS or alveolar RMS.

22. The use of any one of claims 19 to 21, wherein the subject is a human.

23. The use of claim 22, wherein the human is a pediatric human.

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